Panning Separation for Monoclonal Antibody‐Specific T‐Cell Subsets: Effects of Enzymes and Metabolic Inhibitors

Abstract

The mechanisms of “panning,” a simple positive cell separation technique, were examined. Using monoclonal antibodies to “pan” for T cells (T101+, T4+, and T8+), we obtained enriched populations with 90% purity and viability from unfractionated human peripheral blood lymphocytes (PBL). We found that the “panning” method reflects an active process. It is sodium azide inhibitable and independent of the divalent cation concentration. Effective panning does not require capping, patch formation, or DNA synthesis. The cell yields are unaffected by mitomycin‐C or microtubule and microfilament blocking reagents such as concanavalin A, colchicine, and cytochalasin B. This economical technique provides large numbers of functionally intact monoclonal antibody‐specific cells within a relatively short time for further functional and biochemical characterization.

Stimulation of interleukin‐1α and interleukin‐1β release from human monocytes by cyanogen bromide peptides of type ii collagen

“…Patients with OA show several signs similar to those of RA, such as joint destruction. IL-1 has been identified as one of the major factors in joint destruction and lymphocyte activation and has been detected in SF from patients with RA and OA (19)(20)(21).…”

“…Mononuclear cells from 6 healthy individuals and 5 RA patients were obtained from heparinized peripheral blood, by the conventional Ficoll-Hypaque method (18). The mononuclear cell population was then enriched for PBMC by plastic adherence, as described previously (19). About 90% of the adherent cells were monocytes, as detected by esterase staining.…”

Purification of T Cell Populations

“…A panning technique can be used (as described here) to both negatively and positively select a specific sub‐population of cells. It is superior to the antibody/complement lysis technique in that it does not result in the loss of a cell population (Goto, Bluestein, & Zvaifler, ). In addition, it can be used instead of cell sorting of fluorescence‐labeled cells when large numbers of cells are desired.…”

Enhanced cytokine production from human macrophages stimulated by polyethylene particles retrieved from interface membranes after failed total hip arthroplasty

“…Approximately 90% of the adherent cells were monocytes, as detected by esterase staining. 23 These cells were cultured in RPMI 1640 and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) supplemented with penicillin (50 µg/ml) and streptomycin (50 µg/ml) for 24 h, and then incubated at 37°C in 5% CO 2 . The cells were harvested, washed with Gey's balanced salt solution to leave a thin film of fluid on the cells, counted, and dispensed at 1.0 ϫ 10 6 cells/well in a 24-well culture plate (Costar, Cambridge, MA, USA).…”